In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane with the aid of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually at right angles to one another, so that one mirror deflects in the X and the other in the Y direction. The tilting of the mirrors is brought about, for example, using galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors for ascertaining the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation stop), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused onto the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which, by sequential scanning of the specimen, results in a three-dimensional image. Usually a three-dimensional image is obtained by image acquisition in layers.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus scanned one grid point at a time. The measured value must be unequivocally associated with the pertinent scan position so that an image can be generated from the measured data. Preferably, for this purpose the status data of the adjusting elements of the beam deflection device are also continuously measured, or (although this is less accurate) the setpoint control data of the beam deflection device are used.
In a transmitted-light arrangement it is also possible, for example, to detect the fluorescent light, or the transmission of the exciting light, on the condenser side. The detected light beam does not then pass via the scanning mirror to the detector (non-descan configuration). For detection of the fluorescent light in the transmitted-light arrangement, a condenser-side detection stop would be necessary in order to achieve three-dimensional resolution as in the case of the descan configuration described. In the case of two-photon excitation, however, a condenser-side detection stop can be omitted, since the excitation probability depends on the square of the photon density (proportional intensity2), which of course is much greater at the focus than in neighboring regions. The fluorescent light to be detected therefore derives, with high probability, almost exclusively from the focus region; this makes superfluous any further differentiation between fluorescent photons from the focus region and fluorescent photons from the neighboring regions using a stop arrangement.
Arrangements that limit the resolution capability of a confocal scanning microscope are determined, among other factors, by the intensity distribution and spatial extension of the focus of the illuminating light beam. An arrangement for increasing the resolution capability for fluorescence applications is known from PCT/DE/95/00124. In this, the lateral edge regions of the illumination focus volume are illuminated with light of a different wavelength that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, the overall result being an improvement in resolution. This method has become known as STED (stimulated emission depletion).
In microscopy, specimens that, for example, have been prepared with fluorescent dyes are examined. Both one-photon excitation and multi-photon excitation are usual for the excitation of fluorescent dyes. Several different dyes that emit fluorescent light of different wavelengths are often used. The dyes are utilized in such a way that they become attached specifically to specimen constituents.
In fluorescent resonant energy transfer (FRET), fluorescent molecules are excited optically, for example with light of the 488 nm wavelength. The emitted light of these so-called donor molecules, which in this example would have a wavelength of approx. 543 nm, results by way of so-called Foerster transfer in the excitation of other closely adjacent molecules (the acceptor molecules). The excited state of the acceptor molecules decays first into an intermediate state and then into the ground state, emitting light at a wavelength of approx. 570 nm in the present example. In addition to excitation of the acceptor molecules by Foerster transfer, some (undesirable) direct excitation can also occur.
Concentration-independent information about the specimen can be provided by way of the lifetime of the excited state of the fluorescent dyes. The lifetime of the fluorescent dyes depends, inter alia, on the nature and composition of the environment. In cell biology, for example, a measurement of the lifetime of the fluorescent dyes can provide indirect information about the calcium concentration in a specimen region.
Several methods for measuring the lifetime of the excited states of fluorescent dyes have become established. These methods are described exhaustively in Chapters 4 and 5 of the textbook by Joseph R. Lakowicz entitled “Principles of Fluorescence Spectroscopy,” Kluwer Academic/Plenum Publishers, 2nd ed., 1999.
It is usual, for example, to modulate the power level of the exciting light over time in order to draw conclusions, from the phase retardation of the emitted light, as to the lifetime of the excited state. It is also usual to excite the fluorescent dye with short light pulses so that the time delay of the emitting light pulses can be measured electronically.
There are fluorescent dyes that exhibit an excited state lifetime of a few nanoseconds or even in the picosecond range. Such a short lifetime cannot be measured with the known methods, since even with complex electronics it is not possible simultaneously to achieve high time resolution and a good signal-to-noise ratio. In addition, with the aforementioned modulation methods it is extremely difficult to modulate the exciting light at high frequencies. The necessary frequencies (several hundred megahertz or even several gigahertz) cannot be achieved even with acoustooptical modulators.